Re: Setting up indo-1

jphillip@mednet.med.miami.edu
Wed, 12 Apr 95 15:55:38 EST

I thought that I would throw my 2 cents in this indo-1 discussion.
One thing that should be looked at is what you are using to split the
signal. All filters are rated in the "NORMAL" position. Which means
the beam or signal hitting the filter at a 90 degree angle. When you
turn the filter at a 45 degree angle you change the performance of the
filter dramatically. A short pass becomes a band pass etc.. I hope
that this helps. Jim Phillips

______________________________ Reply Separator _________________________________
Subject: Setting up indo-1
Author: G Smith <prsgs@bath.ac.uk> at smtpmed
Date: 4/10/95 2:33 PM

Hello,

I am in the process of setting up an assay for intracellular calcium
mobilisation using indo-1 on a FACS vantage and have run up against a
couple of problems. If anyone could help me, it would be most appreciated.

The UV excited fluorescence signal is split by a 505nm dichroic mirror
and the two fluorescences are collected at 395+/-25nm (Fl4) and
525+/-25nm (Fl5). The dichroic mirror is a curious thing with a sort of
hinge, making it infinitely adjustable. I have aligned the system with
UV beads but get little or no signal in FL5. This may be due to the
spectrum of the beads.

With Indo-1 (3uM) loaded cells, I get a nice signal in FL4, but again no
signal in FL5. Considering that I will be looking for an increase in FL4
and a decrease in Fl5, this is not satisfactory. Is there an established
setting up protocol for this assay? I get nice changes using Fluo-3
alone, but I dont want to use this. I have a lot of noise in Fl5, and
this may be the source of the problem. If anyone has any ideas, Id be
pleased to hear them.

Thanks

Graham
Graham

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